Engineered BAR PAT Enzyme Variants for Reduced Metabolic Interference
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Solution Overview
Problem
The herbicide resistance enzyme bialaphos resistance acetyltransferase (BAR) and phosphinothricin acetyltransferase (PAT) exhibit unpredictable promiscuous activities, leading to interference with plant amino acid metabolism and the formation of non-native metabolites, which complicates the development of genetically modified plants with desired herbicide resistance.
Innovation Solution
Structure-guided engineering of BAR and PAT protein variants with modified acetyltransferase activities against tryptophan or aminoadipate, reducing promiscuous activities while maintaining herbicide deactivation, by introducing specific amino acid substitutions such as N35T, Y92F, and V125T, which are integrated into the genome of transgenic plants to enhance substrate specificity and prevent unwanted metabolite formation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wildtype BAR or PAT protein is expressed in transgenic plants, then herbicide resistance is achieved, but promiscuous acetyltransferase activities interfere with plant amino acid metabolism and form non-native metabolites
Solution Approach 1:
The patent applies local quality by introducing specific amino acid substitutions at targeted positions (N35, Y92, V125) within the BAR/PAT protein structure. These localized changes modify the active site characteristics to reduce promiscuous activity toward plant amino acids while preserving herbicide deactivation capability, thereby resolving the metabolic interference issue without sacrificing herbicide resistance
Solution Approach 2:
The patent employs parameter changes by systematically varying amino acid residues at specific positions in the BAR/PAT protein sequence. Through site-directed mutagenesis, the patent modifies substrate specificity parameters (Km, kcat) for different substrates, achieving a shift in enzymatic preference away from plant amino acids while maintaining effective catalysis toward herbicide targets
2Reliability
If BAR or PAT enzyme is used for herbicide resistance, then glufosinate inactivation is achieved, but substrate specificity is unpredictable and promiscuous activities occur
Solution Approach 1:
The patent improves substrate specificity precision by making targeted local modifications to the enzyme structure. Specific amino acid substitutions at positions N35, Y92, and V125 create a more selective active site that precisely discriminates between herbicide substrates and plant amino acids, eliminating the unpredictable promiscuous activity observed in wildtype enzymes
Solution Approach 2:
The patent utilizes feedback from structural analysis and kinetic characterization of wildtype BAR/PAT enzymes to guide rational protein engineering. By analyzing the relationship between amino acid sequence, three-dimensional structure, and catalytic activity, the patent iteratively optimizes mutant variants with improved substrate specificity while maintaining herbicide deactivation efficiency
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The engineered variants significantly reduce the accumulation of acetyl-aminoadipate and acetyl-tryptophan in transgenic plants, improving herbicide resistance and minimizing metabolic interference, thereby enhancing the predictability and efficacy of herbicide resistance traits in genetically modified crops.
Implementation Method 1
The herbicide-resistance enzyme, generically referred to as bialaphos resistance acetyltransferase (e.g., BAR or PAT) interferes with plant amino acid metabolism through its promiscuous activities. The present invention provides methods for repressing such undesirable activities through structure-guided enzyme engineering.
Data Source
AI summary
Provided herein are engineered bialaphos resistance acetyltransferase variants having a modified acetyltransferase activity against tryptophan or aminoadipate, or both, as compared to a wildtype bialaphos resistance acetyltransferase (e.g., BAR or PAT). Also provided are transgenic plants comprising a bialaphos resistance acetyltransferase variant as well as methods of making such transgenic plants.